Methods for detecting and/or quantifying a concentration of specific bacterial molecules using bacterial biosensors

ABSTRACT

A real-time method employing a portable peptide-containing potentiometric biosensor, can directly detect and/or quantify bacterial spores. Two peptides for specific recognition of B. subtilis and B. anthracis Sterne may be immobilized by a polysiloxane monolayer immobilization (PMI) technique. The sensors translate the biological recognition event into a potential change by detecting, for example, B. subtilis spores in a concentration range of 0.08-7.3×104 CFU/ml. The sensing method exhibited highly selective recognition properties towards Bacillus subtilis spores over other kinds of spores. The selectivity coefficients of the sensors for other kinds of spores are in the range of 0-1.0×10−5. The biosensor method not only has the specificity to distinguish Bacillus subtilis spores in a mixture of B. subtilis and B. thuringiensis (thur.) Kurstaki spores, but also can discriminate between live and dead B. subtilis spores. Furthermore, the sensing method can distinguish a Bacillus subtilis 1A700 from other B. subtilis strain. Assay time may be as low as about 5 minutes for a single test. Rapid identification of B. anthracis Sterne and B. anthracis ΔAmes was also provided.

§0 CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/888,530 (incorporated herein by reference) titled “BACTERIALBIOSENSORS,” filed on Jul. 9, 2004 now abandonded and listing KalleLEVON, Bin Y U and Yanxiu ZHOU as inventors, which claims the benefit ofU.S. Provisional Patent Application Ser. No. 60/486,088 (incorporatedherein by reference), titled “BACTERIAL BIOSENSOR,” filed on Jul. 10,2003 and listing Kalle Levon, Bin Yu, and Yanxiu Zhou as inventors.

§1. BACKGROUND

§1.1 Field of the Invention

This invention relates generally to the field of sensors and inparticular to biosensors specific to biological/chemical agents andbacterium such as Bacillus anthracis.

§1.2 Background of the Invention

The potential use of anthrax, and in particular the spores of Bacillusanthracis (BA) as a weapon of biological terrorism has rekindledinterest in devices and methods for the rapid detection andidentification of biological or chemical agents. Such interest hasbecome particularly acute since the September 11 attacks and theanthrax-by-mail terrorism.

Devices and methods for detecting biological or chemical agents shouldbe rapid, specific, easy to use and transport, and very sensitive sincea single pathogenic organism may be an infectious dose in some cases.Consequently, it is important to assess and begin treatment early forindividuals exposed to such organisms. Additionally, it is equallyimportant to know whether a person exhibiting general symptoms issuffering from, for example, anthrax exposure, or a less serious ailmentfor which totally different (or perhaps antagonistic) treatments areindicated.

§1.3 Related Art

Significant technological progress has been made in the detection andanalysis of biological and chemical agents over the past decade. (See,for example: Ivnitski, D., Abdel-Hamid, I., Atanasov, P., Wilkins, E.Biosensors Bioelectron., 1999, 14, 599-624 and references therein (875);and Iqbal, S. S., Mayo, M. W., Bruno, J. G., Bronk, B. V., Batt, C. A.,Chambers, J. P. Biosensor Bioelectron., 2000, 15, 549-578 and referencestherein (881).)

The outer face of macromolecular biological assemblies like viruses orbacteria includes a proteinaceous capsid, a membrane composed ofglycoproteins and lipids, or a cell wall. Accordingly, they carrycharged or chargeable groups on their outer surface creating an electricdouble layer upon contact with the aqueous phase. (See, for example:Kenndler, E., Blass, D. Trends in Anal. Chem., 2001, 20(10), 543-551;and Lanza, R. P., Langer, R., Chick, W. L. (Eds). When a biologicalrecognition component for bacterial spores, such as a peptides, nucleicacids (See, for example, Park, S.-J, Taton, T. A., Mirkin, C. A.Science, 2002, 295, 1a503-1506.), apatamers (See, for example, Bruno, J.G., Kiel, J. L. Biosensor Bioelectron., 1999, 14, 457-464.), orantibodies (See, for example, Zhou, B., Wirsching, P., Janda, K. D.PNAS, 2002, 99, 5241-5246.) are incorporated in/on a sensing layer of anelectrode, the bacterial spores can be recognized by a biospecificreaction which takes place between the biological recognition componentand bacterial spores—without any pre-concentration or separationprocess.

In particular embodiments, bacterial spores (receptor) and peptide,which is fixed on the surface of substrate, associate in solution toform a peptide-spores biological complex. The residual potential due tocomplementarity between the peptide (the complementary ligand) and thebacterial spores (receptor) with the best possible electrostatic freeenergy change, is equal in magnitude and opposite in sign to the liganddesolvation potential everywhere within the ligand including on theligand surface. (See, for example: Honig, B., Nicholls, A. Science,1995, 268, 1144-1149; Honig, B., Sharp, K., Yang, A.-S. J. Phys. Chem.,1993, 97, 1101-1109; Lee, L.-P., B. Tidor, B. J. Chem. Phys., 1997, 106,8681-8690; Chong, L. T., Dempster, S. E., Hendsch, Z. S., Lee, L-P.,Tidor, B. Protein Sc., 1998, 7, 206-210; and Kangas, E., Tidor, B. J.Chem. Phys., 1998, 109, 7522-7545.) Under the electromotive force(potentiometry), the surface electrostatic potentials of thepeptide-spores complex formed relates to the specific biorecognitionprocess enabling bacterial spores to be identified and detected bypotentiometry.

Many of the technologies developed and/or currently being used however,such as FT-IR spectroscopy, fluorescence spectroscopy, polymerase chainreaction (“PCR”), flow cytometry, impedimetry, UV resonant Ramanspectroscopy, and others, are large, expensive, or requiresophisticated, relatively time-consuming, and often extensive analysisprocedures.

Accordingly, devices and methods which facilitate the accurate, quick,convenient and inexpensive detection of biological or other chemicalagents are of significant scientific and societal interest.

In view of the limitations in the art, a flexible method for selectivelydetecting a wide range of molecules is needed. Additionally, it isdesirable that such methods and devices constructed therefrom beapplicable to the detection of biological or other chemical agents.

Such methods and devices are the subject of the instant invention.

§2. SUMMARY OF THE INVENTION

We have developed methods for using biosensors for the sensing/detectionof biological or chemical agents of significant interest. Our inventivemethods and sensors may involve biological and/or chemical recognitioncomponents (selectors or probes) immobilized on a substrate by using apolymer layer, such as a Polysiloxane Monolayer immobilization (PMI).The PMI method may be used to immobilize the selectors on the substrateby forming a physical bond between the monolayer and the selector (e.g.,by adsorption).

In one embodiment, a monolayer of polysiloxane is polymerized on asubstrate, onto which selector molecules are adsorbed. The resultingimmobilized selector molecules is then used to interact with specificmolecules (targets) within a mixture of molecules. Those specificmolecules are detected and/or quantified via potentiometry.

§3. BRIEF DESCRIPTION OF THE DRAWINGS

Further features and aspects of the present invention may be readilyunderstood from the Drawing in which:

FIG. 1 is a diagram depicting PMI fabrication of a peptide/ODS sensorfor bacterial spores;

FIG. 2 is a diagram depicting the experimental assembly for detecting B.subtilis spores using potentiometry and a peptide/ODS sensor;

FIGS. 3 a and 3 b are graphs showing the potentiometric response ofpeptide/ODS sensor to B. subtilis and Bacillus thurigiensis kursakispores, respectively;

FIG. 4 is a graph (shown as both log concentration and concentration)showing the potentiometric responses of other kinds of spores onpeptide/ODS sensors;

FIG. 5 is a graph (shown as both log concentration and concentration)showing the potentiometric responses of peptide/ODS sensors of B.subtilis strain 1A700 and B. subtilis ATCC 6633, respectively;

FIG. 6 includes a phase contrast microscopic image and fluorescencemicroscopic image of labeled spores.

FIGS. 7(A) and 7(B) are graphs showing the influence of peptideconcentration in the ODS-CHCl₃/CCl₄ deposition solution and the effectof the co-adsorption time on the potentiometric response of peptide/ODSITO sensor;

FIG. 8 is a graph showing potentiometric responses of B. subtilis on ODAITO electrodes with and without peptide and B. thur. Kurstaki with andwithout peptide;

FIGS. 9(A) and 9(B) are graphs showing (A) potentiometric responses ofpeptide/ODA ITO sensor to other kinds of spores and (B) B. subtilis: B.thur. Kurstaki;

FIG. 10 is a graph showing potentiometric responses of peptide for B.anthracis Sterne; B. cereus T; and B. antracis Ames; and

FIGS. 11(A) and 11(B) are graphs showing potentiometric responses ofpolylysine/ODS sensors to B. anthracis Sterne and B. cereus T using(A)log[spores]/ml and (B)[spores]/ml.

§4. DETAILED DESCRIPTION

The following description is presented to enable one skilled in the artto make and use our invention, and is provided in the context of furtherparticular embodiments and methods. The present invention is not limitedto the particular embodiments and methods described.

Polysiloxane monolayer immobilization methods are described in §4.1.Exemplary conditions for the preparation of our inventive biosensor(s),as well as measurements of the properties of experimental bio-sensor(s),are described in §4.2. Finally, exemplary peptide/ODS biosensors aredescribed in §4.3 for B. subtilis spores and B. antracis Sterne.

§4.1 Performing Polysiloxane Monolayer Immobilization

Our inventive immobilization methods generally involves: (1)polymerizing a layer of polysiloxane onto a substrate, and (2) allowingselector molecules to be physically adsorbed to (or coupled via one ormore intermediate elements with), or otherwise immobilized on thesubstrate using the polymer layer. The polymerizing and selectormolecule immobilization may occur substantially concurrently. Theresulting polymer/selector-coated substrate can then be dried andwashed, if necessary.

Polymerizing to obtain a polymer layer with selector moleculesimmobilized in/onto it generally involves soaking a substrate in asuspension of selectors in solvent containing polysiloxane-formingmonomers. After drying and washing, a template may be adsorbed onto thepolysiloxane layer on the support surface and held in place byhydrophobic silanol groups.

The substrate used in PMI acts as a support surface for the polymerlayer. Advantageously, substrate choice(s) largely depend on the methodused for detecting interactions between immobilized selectors and targetmolecules in solution. For example, if potentiometry is used as thedetection method, an electrode may be used as the substrate. Ifmicroscopy is used as the detection method, a microscope slide may beused as the substrate. As a result, and as can be readily appreciated bythose skilled in the art, our inventive methods and sensors constructedaccording thereto accommodate a wide variety of substrates and detectiontechniques (such as microscopy, quartz crystal microbalance, orgravmetric, acoustic, heat generation, conductivity, ion-selectivity,dielectric, magnetic, electrochemical, etc.). Additional sensorysubstrates may include solid-state electronic devices such as diodes ortransistors including insulated gate field effect transistors (IGFETs),metal oxide semiconductor field effect transistors (MOSFETs) and fieldeffect transistors (FETs).

In one preferred embodiment, polymers used in PMI are covalently boundto the substrate's surface. Therefore, the substrate's molecularstructure includes atoms that can bind the polymer monomers on thesubstrate's surface. An exemplary embodiment of our invention uses anindium-tin oxide (ITO) glass electrode as the substrate. As can beappreciated by those skilled in the art, covalent bonding of the polymerto the substrate surface is not required, however, as any type ofmechanical/chemical/electrostatic or other bonding is acceptable, solong as it is sufficiently durable and does not negatively interferewith detection.

Although polysiloxane-forming monomers are described, as can beappreciated by those skilled in the art, other polymer-forming monomersmay be used to generate a polymer layer to immobilize the selectormolecules with respect to the substrate.

With reference now to FIG. 1, there is shown in schematic form a diagramdepicting PMI fabrication of a peptide/ODS sensor for bacterial spores.

More specifically, and as shown in FIG. 1, treating the ITO electrode110 with NaOH replaces OH groups with ONa groups resulting in modifiedITO electrode 120. Such modification generally facilitates covalentbinding of octadecyltrichlorosilane (C₁₈H₃₇SiCl₃) to surface oxygenatoms by displacing Na.

With continued reference to FIG. 1, the modified ITO electrode 120 istreated with a solution containing peptide, C₁₈H₃₇SiCl₃, CHCl₃, andCCl₄, at substantially 0° C., which results in polymer-modified ITOelectrode 130 containing selector peptide 135 adsorbed onto the polymermonolayer.

As can be appreciated, the polymer monolayer functions to hold selectormolecules in place. All -trichlorosilane compounds, such asoctenyltrichlorosilane, cyclohexlmethyltrichlorosilane,bromopropyltrichlorosilane, trichlorosilane, tert-butyltrichlorosilane,ethoxytrichlorosilane, methyltrichlorosilane, pentyltrichlorosilane,etc., which could produce a polysiloxane monolayer are suitable polymersfor PMI. Further, other molecular imprinting polymers (such as thoseprepared with protected amino acid benzyloxycarbonyl-L-tyrosine andeither 2-vinlpyridine, acrylic, (4-vinylphenyl)boronic acid,vinylbenzoic acids, acrylamido-sulfonic acids, amino-methacrylamides,vinlpyridines, vinylimidazoles, acrylamides, vinyl-imminodiacetic acids,or methacrylic acid, or a combination of both, or some otherself-assembly imprinting polymer avoid the need to use the washing stepfor removing the template, and therefore may be considered alternativepolymers for PMI.

Selector molecules are physically adsorbed onto the substrate and heldin place by hydrophobic silanol groups. Selector molecules may be heldto the polymer layer, thereby immobilizing them on the substrate, inother ways. As a result, many biological or chemical materials may beused as selector molecules with our inventive PMI methods. Morespecifically, PMI selectors may include large biomaterials such aspeptides, proteins, enzymes, antibodies, lectins, aptamers or nucleicacids, cells, bacterial tissues, receptors, and other kinds ofbiological and chemical molecular recognition elements. Additionally,selectors may be hydrophobic or hydrophilic, cationic or anionic, andmay be biologically active. Furthermore, other elements and/orstructures may exist between the selector molecule(s) and substrate.(See, e.g., U.S. Provisional Application Serial No. 06/370,502(incorporated herein by reference), titled “DENDRIMER SENSOR FORBAELILLUS SUBTILIS SPORES,” filed on Apr. 5, 2002.

§4.2 Experimental Procedures for Sensor Development and Evaluation

EXPERIMENTAL

Chemicals and Biochemicals

Chloroform and carbon tetrachloride were distilled over CaH₂. Otherchemicals were used without further purification. Peptides werepurchased from Advanced ChemTech, Inc (Louisville, Ky.). Alexa Fluor™488 was obtained from Molecular Probe (Eugene, Oreg.). All aqueoussolution was prepared from water purified using a Millipore System(Resistivity: 18.2 MΩcm) and sterilized by autoclaving.

Bacterial Spores

Bacillus subtilis 1A700 (B. subtilis), B. thuringiensis Kurstaki (B.thur. Kurstaki), B. thuringiensis B8 (B. thur. B8), B. licheniformis, B.globigii, B. anthracis Sterne, B. anthracis ΔAmes, B. cereus T, and B.megaterium ATCC 14581 were grown in our laboratories. B. subtilis ATCC6633 was purchased from Raven Biological Laboratories (Omaha, Me.).

Indium Tin Oxide (ITO) Electrodes and Surface Modification

ITO-coated glass (CG-50IN-CUV), which had a surface resistance of 10Ω/cm² was purchased from Delta Technologies (Stillwater, Minn.). Thesubstrates were cleaned with concentrated nitric acid, 0.02 M sodiumhydroxide, and water, respectively. Before the modification, thesubstrates were dried under a stream of nitrogen. They were subsequently(effective surface area about 1×4 cm²) immersed into a solution of 2:3(v/v) CHCl₃/CCl₄ suspension containing octadecyltrichlorosilane (ODS)(8×10⁻⁴ M) and peptide (0.30 mg/ml) at 0° C. for four minutes. Theelectrode was allowed to stand overnight at −20° C. Then peptide sensorswere washed with water to remove the un-immobilized peptide from thesurface and ready for use. ODS modified electrode without the peptidewas also prepared as a control.

Microscope Imaging

All microscope experiments were performed on a fluorescence Microscope,Nikon Eclipse E600 (Diagnostic Instruments, Inc). 7.2×10⁵ CFU/ml B.subtilis spores were labeled with Alexa Fluor™ 488, and then mixed withthe same amount of unlabeled B. thur. Kurstaki spores. A 1 μL mixture ofthe above spores was added to the surface of peptide/ODS sensors (or thecontrol), and checked under both phase-contrast and fluorescencemicroscope. They were checked under the microscope again afterincubating 10 minutes and washing thoroughly using sterile water toremove non-captured spores.

Electrochemical Measurement

Potentiometric experiments were performed with an Orion 920APotentiometer. All measurements were made in 50 ml of pH 7.4 PBS bufferthermostated to 37° C., in a 100 mL working volume single-compartmentelectrochemical cell (EG&G), equipped with magnetic stirrer. Thetwo-electrode system included an Ag/AgCl (saturated KCl) referenceelectrode and the peptide/ODS/ITO sensor (or the control) workingelectrode. Thereafter, the potential response of the sensor was definedas the difference between the electrode potential with and withoutbacterial spores in solution, i.e., ΔE=E₁−E₀, where E₀ and E₁ are theelectrode potentials before and after the addition of bacterial spores,respectively.

§4.3 Sensors for B. subtilis Spores and B. antracis Sterne

As can now be readily appreciated, our inventive PMI methods ofimmobilizing a selector molecule on a support surface have significantimplications for sensor development. More specifically, PMI may be usedto immobilize a selector on the surface of an electrode, creating asensor that selectively recognizes a molecule that interacts with theselector. We have applied our invention to the fabrication of novelsensors for selectively detecting bacteria, amino acids, and target DNA.

In an exemplary embodiment of our inventive PMI methods, a PMI-modifiedsensor was developed for recognizing Bacillus subtilis spores andBacillus Antracis. Advantageously, and unlike indirect sensors which areresponsive to products of degraded spores (such as those produced bymolecular imprinted polymers, e.g., dipicolinic acid), our inventivePMI-modified sensors are direct sensors in that they are responsive tointact spores.

To fabricate a bacterial sensor, and in particular the B. subtilisspores sensor, an ITO glass electrode may be used as the substrate andPeptide may be used as selectors. Treating the ITO glass electrode withNaOH replaces surface OH groups with ONa groups. The ITO glass electrodemay then be coated by soaking it in a suspension of peptide selectormolecules (˜0.35 mg/ml) and CHCl₃/CCl₄ (a volume ratio of 2:3)containing 0.8 mM ODS for approximately four minutes. Theselector/polymer-coated electrode is then dried overnight and thenwashed with water.

With further reference to FIG. 1, and as shown therein, the peptide 135is immobilized between carbon chains on the ODS monolayer 137 using thisfabrication method. Note further that the template was not washed outwith water since the hydrophobic layer prevents water from approachingthe template. A low polarity medium such as chloroform, however, mayremove the template from the monolayer.

FIG. 2 shows an experimental assembly 200 of B. subtilis spore detectionusing potentiometry and a PMI-modified sensor. The detection assembly200 includes a reference electrode 210 and the B. subtilis spore sensor220, both attached to a potentiometer 230. In this configuration, thereference electrode 210 and sensor 220 are suspended in a solution 240containing B. subtilis spores stirred by a magnetic stirrer 250. Withthis detection assembly 200, the specific interaction betweenimmobilized peptide on the surface of the sensor 220 and B. subtilisspore, produces an electrochemical event which may be measured bypotentiometry. To test the specificity of our inventive peptide/ODSsensor to B. subtilis spores, its detection, versus that of a control(e.g., a blank sensor) detection, of B. subtilis and B. thur. kurstakispores was measured. FIG. 3A shows the sensitivity of the B. subtilispeptide/ODS sensor, and the insensitivity of the blank sensor, to B.subtilis spores. Advantageously, and as expected, neither sensor wassensitive to B. thur. kursaki spores, as shown in FIG. 3B. Similarly,FIG. 4 shows the insensitivity of the B. subtilis peptide/ODS sensor tovarious bacteria spores other than B. subtilis spores. These resultsdemonstrate the high selectivity of the PMI-modified sensor to itstarget molecule.

Of further advantage, our inventive peptide/ODS sensor is sensitive toeven a particular strain of B. subtilis spores. Turning our attentionnow to FIG. 5, it can be seen that our inventive sensor only identifiedthe strain of bacteria (1 A700) for which its immobilized peptides werespecific. A different strain (ATCC 6633) went largely undetected by thesensor.

To further evaluate our inventive method and sensors, two heptapeptideligands that bind species-specifically to spores of selected Bacillussubtilis and anthracis Sterne species were identified by Phase Display(Ph.D.) Ligand Screening System. These peptides were immobilized on thesurface of ITO glass by Polysiloxane Monolayer Immobilization (PMI) in amanner as previously described.

As described before and now stated alternatively, PMI is a novel methodto immobilize chemical or biological molecular recognition elements(probe or ligand) on substrates. Basically, ligand (in this example,peptide) and the silylating agent (ODS) were co-adsorbed on the polarsolid surface of the ITO glass plates, the ligand was incorporated intoa polysiloxane monolayer by forming a hydrophobic layer of polymerizedorganosiloxane groups around the molecule recognition elements asdepicted in FIG. 1.

The chemical or biological recognition ligand was dissolved or suspendedin a low polarity medium during immobilization. The ligand could not beremoved with water since the hydrophobic polymer monolayer preventswater from approaching the ligands, even though most of the ligands arewater soluble. This may be due to the hydrophobic layer preventing waterapproach to peptide, which was surrounded by aliphatic chain. Therefore,the resulting immobilized ligand may then be used to interact withspecific chemical or biological targets within a mixture of molecules,thereby enabling specific analyte to be detected and/or quantified.

To find the optimal biorecognition conditions, the influences of thepeptide concentration in the deposition solution and co-adsorption timeduring the fabrication of the sensors were examined. When theconcentrations of peptide concentration ranging from 0.1 to 0.55 mg/mlin CHCl₃/CCl₄-ODS solution, the potentiometric output of those sensorsis shown in FIG. 7(A).

When the concentrations of peptide is 0.30 mg/ml, the resulted sensorproduced the highest potential responses to 1.3×10⁻⁵ CFU/ml B. subtilisspores in 1.0×10⁻² M phosphate buffer-1.5×10⁻¹ M NaCl (PBS buffer, pH7.4). After the immersion of the pretreated ITO-coated plates into thesolution containing ODS and peptide for time periods varying from 1 to 7minutes, the potential responses of these electrodes were observed at B.subtilis spores concentrations ranging from 0.08 to 2.0×10⁵ CFU/ml B.subtilis spores. The peak response was observed at an immersion time ofapproximately 4 minutes, as shown in FIG. 7(B).

The influence of incubation temperature was also investigated since itcannot be guaranteed that 37° C. will be the temperature when the sensoris used in field. At 28.5° C., the signal remains 97.7% of that producedat 37° C., and 48.6% of that produced at 23° C. Incubation temperaturehas a marked influence on biorecognition process and those devices canstill be used at around 25° C.

In FIG. 8, curve (----) is control (without peptide) towards B.subtilis. As the concentration of B. subtilis increases, potentialoutput decreases. When the concentration of B. subtilis is 500 CFU/ml,the potential found with the control does not decrease further, andstabilizes. This characteristic indicates that B. subtilis has strongnegative charge. However, when the peptide is immobilized in theselective layer of the sensor, the results (FIG. 8, curve (——))demonstrated potential change in a direction opposite to that withoutpeptide (FIG. 8, curve (----)). The potential response of the sensor tothe B. subtilis spores in a PBS suspension was linear in theconcentration range of 0.08-7.3×10⁴ CFU/ml and the limit of detection(LOD) was 0.08 CFU/ml.

The distinctive difference in the potential outputs can be observedbefore and after immobilizing the peptide onto the active surface ofITO. This phenomenon indicates that peptide was successfully linked tothe ITO surface. Peptide ligand (7 amino acids long) containing theconsensus sequence NHFLP binds tightly, and species specifically, tospores of B. subtilis and forms a complex. This peptide—B. subtiliscomplex carries positive charge, which is totally different from thespores themselves (FIG. 8, curve (----)).

B. thur. Kurstaki spores were used to evaluate the binding of other kindof spores to the peptide/ODS surface. Curve (

) and curve (

) in FIG. 8 were potential response of a control (without peptide)sensor and a peptide sensor (with peptide) to B. thur. Kurstaki spores,respectively. Results obtained show that negative charge of B. thur.Kurstaki spores did not change after they encountered peptide. In otherwords, the peptide did not have any significant affinity for B. thur.Kurstaki spores.

In the case of B. subtilis, the peptide ligand appears to mimic thebinding of the SpsC protein. This protein apparently binds to thesurface of the forespore and may be required for the synthesis ofsurface polysaccarides late in the spore development. Polysaccaridedeposition apparently causes B. subtilis spores to be hydrophilic.Genetic inactivation of the operon encoding SpsC causes B. subtilisspores to become hydrophobic much like spores of B. anthracis.Accordingly, B. subtilis Asps spores may provide an improved simultantfor spores of B. anthracis.

Optical microscopy was also used to evaluate the binding of bacterialspores to the surface-confined peptides. A representative image ofpeptide sensor after reaction with 1.44×10⁶ mixture of same amount of B.subtilis and B. thur. Kurstaki spores on the surface is shown in FIG. 6.Both spores were observed under phase contrast. However, only B.subtilis spores could be seen under fluorescence as they are labeledwith fluorescence reagent, Alexa 488. Images obtained at severaldifferent locations on the surface show that the selectivity of theimmobilized peptide to B. subtilis is 98%, due to the very similarpictures of the same sensor under phase contrast and fluorescencemicroscope.

As can be appreciated, effective testing of bacteria requires methods ofanalysis that meet a number of challenging criteria. Selectivity andtime of analysis are important characteristics related to the usefulnessof microbiological testing. The biosensor system should have thespecificity to distinguish the target bacteria or bacterial spores fromothers. FIG. 9(A) shows the potentiometric output in pH 7.4 PBS bufferupon treatment of sensing interface with same amount (2.5×10⁵ CFU/ml)but different kinds of bacterial spores. The data of potentiometricmeasurement in the inset figure in FIG. 9 (A) can be simulated withNicolsky-Eisenman Equation:E=E _(B.subtilis) ⁰ +s log([B. subtilis]+K _(B.subtilis,j) ^(POT) [a_(j) ^(z) ^(j) ])  (1)where E_(B.subtilis) and E_(B.subtilis) ⁰ are the potential of thesensor and the standard electrode potential, respectively, s is theslope. K_(B.subtilis,j) ^(POT) (j, interference of charge z_(j)) wasobtained for peptide biosensor based on simulation results of theexperimental data shown in FIG. 9(A), and shown in Table 1.

TABLE 1 Potentiometric selectivity coefficients B. thur. B. megateriumB. anthracis Spores Kurstaki B. licheniformis B. thur. B8 B. globigiiATCC 14581 Sterne K_(B. subtilis,j) ^(POT) 0 0 1.0 × 10⁻⁵ 0 2.5 × 10⁻⁶12.5 × 10⁻⁹Besides B. subtilis, none of those bacterial spores produced any falsepositive potentiometric response, implying that the peptide is specificfor B. subtilis.

The high selectivity of our inventive biosensor also reflects in theidentification of B. subtilis in the presence of other bacterial spores,e.g., B. thur. Kurstaki spores (FIG. 9(B) (blank line)). The resultsindicate that the sensor has almost the same potential response in thepresence of the equal amounts of the B. thur. Kurstaki at concentrationranges from 0.08 to 9000 CFU/ml. No substantial potential difference wasobserved with and without B. thur. Kurstaki, which demonstrates a highdegree of selectivity. The biosensor could distinguish B. subtilis fromother strains of the same species, such as B. subtilis ATCC 6633 (FIG.9(B)(— —— ), which strain is different from B. subtilis 1A700. In thepresence of 1.3×10⁵ CFU/ml, the biosensor gives 55.8 mV to B. subtilis1A700 and only 4.6 mV for B. subtilis ATCC 6633 demonstrating highlyselective of the peptide/ODS sensor. Advantageously, even live and deadspores can be distinguished from one another. As demonstrated in FIG.9(B),(—— ——), as B. subtilis 1A700 gave no more positive potentialresponse after autoclaved.

Following the same facile modification of patterning and assayprocedures, another kind of inventive peptide/ODS biosensor wasdeveloped for the detection of B. anthracis Sterne spores.

As was the case with B. subtilis, this peptide sequence binds tightlyand species specifically to spores of B. anthracis Sterne solutionranging in concentration from 0.8 to 2.5×10⁷ CFU/ml and forms a positivepotential response complex.

With reference now to FIG. 10, there is shown a graph depictingpotentiometric responses of sensors for anthrax spores constructedaccording to our inventive teachings for ODI/peptide for B. anthracisSterne; B. cereus T; and B. antracis Ames. As can be readilyappreciated, the response is quite specific to the anthrax spores.

As can be seen by inspection of FIG. 10, B. cereus T (FIG. 10 (□)),which is very similar to B. anthracis Sterne, is undetected by ourODI/peptide sensor. And while both kinds of spores demonstrated bindingby FACS analysis, potentiometric measurement advantageously provides thebias for the discrimination between B. anthracis Sterne and B. cereus T.

Additionally, the same sensor was employed to identify B. anthracisΔAmes, which should have more affinity for this peptide than B.anthracis Sterne, as shown in FIG. 10 (♦)). As is shown in FIG. 10, whenthe concentration of bacterial spores <10⁷ CFU/ml, B. anthracis Sterneproduced a highly potentiometric output than B. anthracis ΔAmes, butwhen >10⁷ CFU/ml, the sensor yielded higher affinity of the peptide forB. anthracis ΔAmes than B. anthracis Sterne.

Lastly, and as mentioned earlier, there are a number of chemical orbiological molecular recognition elements (probe or ligand) that may beimmobilized on suitable substrates and used to providechemical/biological specificity to a sensor. FIGS. 11(A) and 11(B) showgraphs of such specific sensors.

As noted before, ligand (in this example, polylysine) and the silylatingagent (ODS) were employed. The resulting immobilized ligand (polylysine)was then be used to interact with specific chemical or biologicaltargets, thereby enabling specific analyte to be detected and/orquantified. With reference to FIGS. 11(A) and 11(B), there is shownpotentiometric measurements of B. anthracis Sterne and B. cereus T shownin both log[spores]/ml and [spores]/ml. As can be readily appreciated bythose skilled in the art, our inventive methods and resulting devicesare capable of providing a wide array of specific sensors and mayinclude, for example, synthetic molecules that exhibit specific binding.

§5. Conclusions

Sensors and methods of fabricating same for the detection andidentification of biological or other chemical agents, and in particularbacteria, so far are characterized by a lengthy analysis time. The assaytime is usually on the order of several tens of minutes to severalhours, even days. For our inventive peptide/ODS sensor, the timerequired to obtain equilibrium and incubation is five (5) minutes for asingle test. Consequently, the biospecific reaction between, forexample, B. subtilis spores and peptide is directly determined in realtime by measuring the potentiometric changes induced by the complexformation between peptide and B. subtilis spores. Our inventive sensorsand method of making same offers the potential to speed up the detectionof anthrax and other pathogenic bacteria.

Additional problems facing the production of biosensors for directdetection of bacterial spores include the sensitivity of assay in realsamples, long lifetime of the sensor and non-specific adsorption. Withour inventive sensors, the limit of detection (LOD) is on the order of 8CFU/100 ml, as described above, which is much improved compare toexisted techniques.

Still further, after being stored at −20° C. in a freezer for severalmonths, the response still remained to 40% of its initial magnitude,demonstrating the long lifetime of our inventive sensors.

Various modifications to the disclosed embodiments and methods will beapparent to those skilled in the art, and the general principles setforth may be applied to other embodiments, methods and applications.Thus, the present invention is not intended to be limited to theembodiments and methods. For example, although various embodiments ofthe invention were described in the context of sensing biologicalmaterial, the teachings of the present invention can be applied tosensing other substances, such as chemicals. Additionally, our inventiveteachings should be read to include a broad array of immobilizationtechniques, in which a bio-active material is brought together withsufficient monomer, such that when the monomer polymerizes on a suitablesubstrate, the bio-active material becomes sufficiently immobilizedwithin the polymer to create a bio-active layer(s) on the surface of thesubstrate. When the substrate is a suitable sensor, the sensor becomes abio-active sensor exhibiting the specificity of the bio-active material.

What is claimed is:
 1. A method for determining whether or not asolution includes specific bacterial molecules, the method comprising:a) providing a reference electrode in the solution; b) providing aworking electrode in the solution, the working electrode including anelectrode substrate having a layer of polymer applied thereto, andbacterial specific selector molecules, wherein the working electrode isconstructed by exposing the electrode substrate to a suitable monomer inthe presence of the bacterial specific selector molecules such that whenthe monomer is polymerized, the selector molecules are substantiallyimmobilized thereto, and wherein the working electrode is constructed inaccordance with an optimal concentration of the bacterial specificselector molecules and an optimal immersion time that are predeterminedto produce a peak potential response during a testing, the optimalconcentration being approximately 0.3 mg/ml and the optimal immersiontime being approximately 4 minutes, and wherein the layer of polymer isa monolayer of Polysiloxane, and wherein if the selector molecules areexposed to the specific bacterial molecules, a change in electricalpotential is caused; c) determining a potential difference between thereference electrode and the working electrode; and d) determiningwhether or not the solution includes the specific bacterial moleculesusing the determined potential difference.
 2. The method of claim 1wherein the bacterial specific selector molecules are peptides.
 3. Themethod of claim 2 wherein the electrode substrate includesIndium-Tin-Oxide (ITO) glass.
 4. The method of claim 3 wherein the ITOglass is treated with Sodium Hydroxide (NaOH).
 5. The method of claim 1wherein the bacterial specific selector molecules are selected from thegroup consisting of: peptides, proteins, enzymes, antibodies, lectin,nucleic acids, and bacterial tissue.
 6. The method of claim 1 whereinthe bacterial specific selector molecules are hydrophilic.
 7. The methodof claim 1 wherein the bacterial specific selector molecules arehydrophobic.
 8. The method of claim 1 wherein the bacterial specificselector molecules are peptides specific to intact spores of BacillusSubtilis.
 9. The method of claim 1 wherein the bacterial specificselector molecules are peptides specific to intact spores of Bacillusanthracis.
 10. The method of claim 1 wherein the bacterial specificselector molecules are peptides specific to intact spores of Bacillusanthracis Sterne.
 11. The method of claim 1 wherein the referenceelectrode and the working electrode are provided on a solid-stateelectrode device.
 12. The method of claim 1 wherein the bacterialspecific selector molecules are synthetic molecules that exhibitselective binding to specific bacterium.
 13. The method of claim 1wherein the bacterial specific selector molecules include polylysine.14. The method of claim 1 wherein the bacterial specific selectormolecules are held to the layer of polymer by hydrophobic silanolgroups.
 15. The method of claim 1 wherein a potential difference betweenthe reference electrode and the working electrode can be determined fora solution a concentration of the specific bacterial molecules of 8 CFU/100 ml.
 16. The method of claim 8 wherein the potential differencebetween the reference electrode and the working electrode is linear forBacillus Subtilis in a concentration range of 0.08-7.3×10⁴ CFU/ml. 17.The method of claim 1 wherein the bacterial specific selector moleculesare hepta peptide ligand selector molecules.
 18. The method of claim 1wherein the acts of (1) determining a potential difference between thereference electrode and the working electrode and (2) determiningwhether or not the solution includes the specific bacterial moleculesusing the determined potential difference are done in real time.